Viruses are used in many scientific applications, especially in the development of prophylactics, therapeutics, and diagnostics. For these purposes, viruses are often subjected to genetic engineering. In vivo engineering requires a tractable host organism and can often take weeks to months to create modified viruses and viral vectors (Levin and Bull, Nat Rev Microbiol., 2004 February; 2(2):166-73, incorporated herein by reference). Additionally, there are toxicity concerns inherently associated with the manipulation of many viral genomes in cells. Efforts to develop methods for in vitro genetic engineering of large viral genomes have thus far been constrained by the availability of unique restriction enzyme target sequences and the low efficiencies obtained for genome digestion and subsequent recombinant assembly. Furthermore, many genetic engineering efforts are thwarted by incorrectly predicted viral genomic termini. For example, publicly available PB1-like viral genomes incorrectly place the end sequences in the middle of the genome, an often occurring error using current sequencing and in silico genome assembly methods (Ceyssens et al., Environ Mibrobiol. 2009 November; 11(11):2874-83).
There remains a need for the rapid genetic engineering of viral genomes, especially for viruses infecting non-genetically tractable hosts. The present disclosure utilizes in vitro Cas9 mediated digestion and assembly to site specifically engineer whole viral genomes. This method drastically increases the precision, simplicity and speed at which viral genomes can be genetically modified. Further, this technique overcomes the well-established difficulty of manipulating often toxic virulent viral genomes inside native and heterologous host cells. Utilizing the disclosed in vitro engineering method also enables identification of correct viral genomic ends, which facilitates subsequent engineering via the present disclosure.
In vitro error correction is an invaluable technique for generating desired sequences following cloning or assembly techniques. Standard error correction methods are PCR-based, which has two inherent problems: 1) PCR can introduce additional unwanted mutations into the nucleic acid and 2) PCR, in this context, has a size restriction of approximated 5 kb before it becomes increasingly error prone (Quick Change site-directed mutagenesis kit manual, New England Biolabs, USA). Therefore, standard PCR-based error correction methods cannot reliably be performed on plasmids larger than 5 kb, either as a result of additional PCR-generated mutations or a failure to amplify the complete template.